Automate BRD4 Bromodomain TR-FRET Assays For HTS Applications

By Pete Brescia, Brad Larson, and Peter Banks, BioTek Instruments, Inc., and D. Bochar and L. Blazer, Cayman Chemical Co.

Here we demonstrate the combination of a homogeneous, time-resolved fluorescence resonance energy transfer (TR-FRET) assay with automated instrumentation and a high-throughput screening (HTS) multi-mode microplate reader that monitors BRD4 bromodomain 1 activity. This system provides rapid characterization in a  high-throughput format as demonstrated using a small natural product library under primary screening conditions  against BRD4 bromodomain 1 (human amino acids 49-170).

Post-translational histone modifications are critical for modulation of chromatin structure by direct DNA interaction and indirect molecular interactions with recruited nuclear proteins. Chromatin structure modulation is proven necessary for gene regulation, repair and cell cycle progression and other processes. Bromodomains are small nuclear proteins that selectively bind to acetylated lysine residues of histone proteins. Once bound, these bromodomains recruit protein complexes involved in chromatin structure regulation, and thus play a role in gene expression. The bromodomain and extra-terminal (BET) protein family includes BRD2, BRD3, BRD4 and BRDT. These proteins all play key roles in diverse cellular processes, such as inflammatory gene expression, mitosis and viral/host interactions, by linking acetylation marks to transcriptional regulation at promoters. Small molecule inhibitors disrupt bromodomain/ histone interactions, holding promise as potential therapeutics for human disease.